Melanoma is the 5th most common type of cancer, and MAPK pathway activation is central to the disease, making it a key druggable target. However, despite the establishment of targeted inhibitors with profound activity in patients, their ultimate effectiveness is limited as resistance quickly develops. The development of drug resistance is a significant hurdle in the treatment of cancer and long-term survival of patients. We have observed that the error-prone Y-family DNA polymerase kappa (pol?) is upregulated during treatment of a melanoma cell line with a BRAF inhibitor and that levels return to normal upon the development of resistance. This has led us to hypothesize that pol? is increasing tumor heterogeneity via stress-induced mutagenesis (SIM). Stress-induced mutagenesis allows cells to temporarily increase their mutation rates when they are maladapted to their environment. Stochastically, a small number of these mutations will be beneficial and promote survival. Many examples of SIM exist, including upregulation of DinB, a polymerase with homology to pol?, by E. coli in response to starvation. This proposal has two specific aims. In Aim 1, we will investigate the mechanism by which BRAF inhibition results in increased pol? expression, specifically focusing on the responsible transcription factors and the role of the DNA damage repair (DDR) pathway. We are in the process of creating a transgenic reporter cell line expressing the fluorophore tdtomato under the control of the pol? promoter. We will use this construct to study the effects of knocking down candidate transcription factors and/or mutating key residues for their binding. We will also study the contribution of the DDR pathway by assaying for DNA damage and/or activation of the DDR components upon treatment with a BRAF inhibitor. In Aim 2, we will use in vitro and in vivo studies to investigate the functional roe of pol? in the development of drug resistance and stress-induced mutagenesis in cancer. For these studies, we have generated a doxycycline-inducible pol? construct for use in our human melanoma cell culture model and zebrafish model of melanoma. Effects on the mutation rate after pol? induction will be determined using exome sequencing. In addition, cells and tumors will be exposed to a pulse of pol? and then assayed for resistance to a BRAF inhibitor. Together these studies should give us a better understanding of the mechanism by which pol? is upregulated and the functional consequences of this increased expression.